MicroRNAs: A Link between Mammary Gland Development and Breast Cancer

Breast cancer is among the most common cancers in women, second to skin cancer. Mammary gland development can influence breast cancer development in later life. Processes such as proliferation, invasion, and migration during mammary gland development can often mirror processes found in breast cancer. MicroRNAs (miRNAs), small, non-coding RNAs, can repress post-transcriptional RNA expression and can regulate up to 80% of all genes. Expression of miRNAs play a key role in mammary gland development, and aberrant expression can initiate or promote breast cancer. Here, we review the role of miRNAs in mammary development and breast cancer, and potential parallel roles. A total of 32 miRNAs were found to be expressed in both mammary gland development and breast cancer. These miRNAs are involved in proliferation, metastasis, invasion, and apoptosis in both processes. Some miRNAs were found to have contradictory roles, possibly due to their ability to target many genes at once. Investigation of miRNAs and their role in mammary gland development may inform about their role in breast cancer. In particular, by studying miRNA in development, mechanisms and potential targets for breast cancer treatment may be elucidated.


Introduction
Breast cancer is among the most common cancers in women, second to skin cancer [1]. Treatment of breast cancer is complex and often depends on the subtype, as developed by Perou et al. [2]. Currently, breast cancer subtypes have evolved to include genomic, proliferative, and immune cell markers [3]. Early breast cancer detection and prevention is commonly associated with BRCA1 or BRCA2 mutations [4], but other structures in the mammary gland may have predictive value as well. For instance, during puberty, terminal end bud (TEB) structures develop, which are the most common sites for tumorigenesis. In later life, these structures differentiate into alveolar buds (AB), which have a reduced risk of de novo tumorigenesis. It has been shown that the number of TEB structures in early life is modifiable, for example via dietary interventions, leading to reduced breast cancer risk later in life [5]. Furthermore, processes critical to normal mammary gland function such as apoptosis, proliferation, and invasion are often altered, leading to breast cancer formation. In fact, a subset of microRNAs (miRNAs) with expression varying through stages of development (juvenile, puberty, mature virgin, gestation, lactation, early involution, and late involution) were also found to be associated with the luminal A breast cancer subtype [6]. This suggests that parallel roles for oncogenic and anti-oncogenic miRNAs may exist during development and during breast cancer. MiRNAs are short, noncoding RNAs, and are required for normal development across species. In the mammary gland, aberrant expression of miRNAs can alter critical functional and developmental processes leading to the development of breast cancer [7]. Therefore, miRNA expression in the mammary gland is a promising clinical biomarker. MiRNAs are also a promising therapeutic target for breast cancer. Investigation of the role of miRNAs in mammary gland , Intrinsic breast cancer subtypes, markers, and characteristics from best prognosis to worst prognosis. Cancer type incidence are as per [1,18] Created with BioRender.com.

MicroRNAs
MiRNAs are short, non-coding 19-22 nucleotide RNAs which post-transcriptionally regulate gene expression [24]. MiRNAs are transcribed by RNA polymerase II in the nucleus, after which Drosha-a ribonuclease type III (RNAse III)-and DGCR8 binds and preprocesses the pre-miRNA. The pre-miRNA (~70 nt long) is transported into the cytoplasm where another RNAse III Dicer cuts pre-miRNA into mature miRNA approximately 22 nt long. This mature miRNA associates with argonaute protein (AGO) to form a RNA-induced silencing complex (RISC) where the miRNA can bind to the 3′ UTR of a mature mRNA, among other possible binding sites, as previously reviewed [24]. One miRNA can target multiple mRNAs, and one mRNA can be regulated by multiple miR-NAs, allowing for the coordinated regulation of several mRNA targets and, by extension, proteins and pathways. In development, miRNAs are critical in regulating developmental timing and progenitor cell fate, and these roles have been extensively reviewed by De-Veale et al. [25]. Dysregulation of miRNAs involved in developmental or homeostatic processes can lead to breast cancer; the role of miRNAs involved in breast cancer develop- , Intrinsic breast cancer subtypes, markers, and characteristics from best prognosis to worst prognosis. Cancer type incidence are as per [1,9] Created with BioRender.com.
Embryo: The mammary gland is separated into the ectoderm and mesoderm, with development beginning around day 10, where it appears as an epithelial bud. The ectoderm forms multiple layers which develop into one pair of placodes (five pairs in mice, six pairs in rats) by day 12 [8]. Around day 13, epithelial cell fate begins to be guided by inductive signals from the mesenchyme. This signaling guides the patterning of the placodes and the positioning of extracellular matrix components. At day 14, the epithelium of the placodes expands and invades the preadipocytes, which are thought to emerge from the mesenchyme [10]. The epithelial cells reach these cells and begin branching, leading to an early ductal system. The ductal lumen forms from day 16 to day 18. Finally, the sexual delineation and nipples are formed. During embryogenesis, the epithelial-mesenchymal in-(EMT) may increase the likelihood of metastasis and expression of breast cancer stem-celllike properties [19]. Anti-oncogenic factors include early age at pregnancy and factors associated with pregnancy such as high doses of estrogen and progesterone, differentiation of TEB structures, and involution [12].
Breast cancer largely arises in epithelial cells in terminal ductal lobular units [20], or undifferentiated TEB in rodents [21]. The TEB, when in the process of differentiating, also has the potential for carcinogenesis. The differentiation of TEB into AB reduces the potential for the development of malignant tumours. This has been demonstrated with 7,12-dimethylbenz[a]anthracene (DMBA) in vivo [22,23], where administration of DMBA at 20 days of age resulted in death of 36/42 rats. Tumour incidence increased with age until 46 days, after which there was a decrease in malignant tumour incidence and increase in benign tumours.

MicroRNAs
MiRNAs are short, non-coding 19-22 nucleotide RNAs which post-transcriptionally regulate gene expression [24]. MiRNAs are transcribed by RNA polymerase II in the nucleus, after which Drosha-a ribonuclease type III (RNAse III)-and DGCR8 binds and preprocesses the pre-miRNA. The pre-miRNA (~70 nt long) is transported into the cytoplasm where another RNAse III Dicer cuts pre-miRNA into mature miRNA approximately 22 nt long. This mature miRNA associates with argonaute protein (AGO) to form a RNAinduced silencing complex (RISC) where the miRNA can bind to the 3 UTR of a mature mRNA, among other possible binding sites, as previously reviewed [24]. One miRNA can target multiple mRNAs, and one mRNA can be regulated by multiple miRNAs, allowing for the coordinated regulation of several mRNA targets and, by extension, proteins and pathways. In development, miRNAs are critical in regulating developmental timing and progenitor cell fate, and these roles have been extensively reviewed by DeVeale et al. [25]. Dysregulation of miRNAs involved in developmental or homeostatic processes can lead to breast cancer; the role of miRNAs involved in breast cancer development, treatment, diagnosis, prognosis, and in exosomes have been previously reviewed [26][27][28]. Here, we discuss miRNAs which can be found in both mammary gland development and breast cancer in the context of functions they may have in common.

MiRNAs in Mammary Gland Development and Breast Cancer
This section discusses characteristics common to development and cancer of the breast, such as EMT, stem-cell characteristics, proliferative ability, angiogenesis, apoptosis, and epigenetic regulation. Previous reviews on miRNAs in mammary gland development and breast cancer focus on subsets of developmental stages [29,30]. Here, we seek to provide a comprehensive updated review of miRNAs in mammary gland development and breast cancer. The literature was searched for miRNAs in mammary gland development and breast cancer across species; 32 miRNAs were found to be shared in both processes, as specifically discussed below and illustrated in Figure 2. 424(322)/503 family increases in involution, and knockout of this miRNA reduces acini destruction and apoptosis [73]. Thus, the miR-424(322)/503 family plays an important role in apoptosis and has a tumour-suppressive effect in breast cancer (Table 1). MiR-424-5p has been shown to reduce chemoresistance and decrease breast cancer proliferation by inducing apoptosis and targeting the PI3K/Akt/mTOR pathway [74,75]. By investigating the role of miRNAs in apoptosis, particularly in involution, future studies may identify dysregulated apoptotic miRNAs as potential therapeutic targets.

EMT
Embryogenesis in the mammary gland is marked by differentiation, migration, and invasion facilitated by a progenitor cell population and epithelial-mesenchymal reciprocity [31]. One hallmark of breast cancer is the reactivation of embryonic programming, particularly with respect to stem-like breast cancer traits and EMT, leading to oncogenesis and metastasis. Embryonic development relies on a delicate interplay between regulatory molecules, such as miRNAs, and expression of genes or transcription factors, and aberrant expression of miRNAs at any stage can dysregulate gene expression leading to breast cancer invasion or metastasis. EMT traits also increase breast cancer chemoresistance by inhibiting apoptosis and increasing chemoresistance-related gene expression [32]. Previous work on embryonic programs in breast cancer has largely focused on parallels in gene-based signatures and pathways [33], and here we will explore this link through miRNA expression. During embryogenesis, miR-137 was found to be highly expressed in the mammary gland, a 30-fold increase compared to surrounding regions [34]. Lentiviral overexpression of miR-137 in ICR mouse embryos led to the thickening of mammary epithelium and inhibition of invasion of the mammary epithelial bud, while MDA-MB-231 tumour formation in vivo was inhibited by miR-137 overexpression [34]. The restriction of mammary bud invasion and tumour formation is consistent with a decreased ability for EMT, where EMT loss may reduce the ability of mammary cells to lose polarity and migrate/invade the underlying fat pad [35]. Indeed, several studies have shown a suppression of invasion, migration, or EMT from miR-137 overexpression [36][37][38][39], leading to reduced metastatic potential and chemoresistance. For example, Lee et al. found that miR-137 was downregulated in triple-negative MDA-MB-231 and Hs578T breast cancer cell lines, and targeted the 3 UTR of Del1, encoding for a protein which is abundantly expressed in plasma of breast cancer patients [38]. MiR-137 has been found to increase protein expression of epithelial marker E-cadherin, and reduce mesenchymal markers N-cadherin and vimentin by modulating DUSP4, leading to reduced doxorubicin resistance [37]. The overexpression of miR-137 in triple-negative MDA-MB-231 cells decreased migration and invasion, pointing to a tumour suppressor role of miR-137 in breast cancer. Thus, an embryogenesis-related miRNA is dysregulated in breast cancer EMT. As shown in Table 1, only one other miR (miR-206) that is altered in mammary gland embryogenesis has been studied in breast cancer, highlighting a research gap for further investigation.

Stemness Characteristics
A stem cell population is present in the mammary gland at all stages of development. Multipotent stem cells are present in the embryo, and, as the development stage progresses, the progenitor cell population becomes restricted to bipotent and unipotent progenitors. These progenitor cells are most active in puberty and pregnancy, when there is rapid proliferation and differentiation of ductal structures or alveologenesis. Some "stem cell" genes have been found to be exclusively expressed in the outer cap cell layer of the TEB in puberty, while others are also present in the basal cell population. The multipotent mammary stem cells have a gene signature which resembles claudin-low and basal-like breast cancer types, characterized by increased aggressiveness and metastasis [40]. Due to their plasticity, stem-cell-like breast cancer cells, identified by CD44 + /CD24 −/low markers, have an increased proclivity for invasion, treatment resistance, and cancer recurrence [19].
During embryogenesis, miR-206 was found to be highly expressed in the mesenchyme at day 11.5. At day 13.5, miR-206 expression was reduced in the mesenchymal layers and localized to the mammary fat pad. Overexpression of miR-206 led to severe stunting of mammary bud formation, indicating that miR-206 may abrogate mesenchymal differentiation [41]. MiR-206 has also been found to be highly expressed in pregnancy, indicating it may play a role in restricting lineage/differentiating progenitor cells [42]. In triple-negative breast cancer cell lines, miR-206 mimics reduced the CD44 + /CD24 −/low cell population. In breast cancer stem cells, miR-206 has been found to inhibit proliferation, metastasis, and increase apoptosis [43]. However, miR-206 has also been found to promote MDA-MB-231 and SK-BR-3 in vitro invasion, migration, and proliferation as well as tumour size in vivo [44]. The differences in response may be due to differing cell types or heterogeneity of miRNA targets, and further investigation is necessary.
Female puberty is characterized as a period of ductal growth, invasion, and proliferation, led by differentiation of bipotent and unipotent stem cells. The nulliparous TEB contains a unique progenitor cell population, with alveolar progenitors, basal progenitors, and cap cells able to differentiate into myoepithelial cells [13]. MiRNAs that are enriched/depleted in TEBs or alter TEB morphology may give information as to their role in breast cancer. For example, miR-34a expression has been found to be low in a stem cell (PKH26+ or CD61+/CD49+) population, but increased along a luminal differentiation route. MiR-34a depletion increased TEB size through an increase in the progenitor cell population [45]. Based on the effect of miR-34a on TEB development, it could be predicted that miR-34a confers a tumour-suppressive effect through the inhibition of a highly proliferative, cancer stem-cell-like phenotype. In several studies, miR-34a has been found to reduce cancer stemness by targeting genes such as CD24, NOTCH1, NOTCH4, HDAC1, or HDAC7. It has also been found to suppress tumour proliferation, EMT markers, and reduce chemoresistance.
Changes during pregnancy may result in a marked reduction in the proliferative population of cells in the mammary gland, leading to reduced breast cancer risk. In a comparison of Holstein cows during mid-pregnancy and during mid-lactation, miR-139 was found to be upregulated in pregnancy [46]. During pregnancy, there is a population of luminal progenitor cells that gradually differentiates as pregnancy advances, ending with fully differentiated cells in lactation [12]. MiR-139 mimics downregulated members of the IGF1R and PI3K/Akt pathways through binding of the GHR 3 UTR. In breast cancer, miR-139 reduced stemness through modulation of the PI3K/Akt pathway by targeting CXCR4. In vivo, miR-139 transfection in MDA-MB-231 cells reduced lung metastatic nodule development. Thus, miRNAs upregulated in pregnancy compared to lactation may be involved in directing the fate of progenitor cells.
For the maintenance of the progenitor population in the mammary gland, miR-205 is required. MiR-205 is expressed predominantly in basal cells through all development stages. It is also expressed in the mammary stem-cell population, although expression is markedly reduced mid-to-late lactation and in involution. In histological sections at the involution of the mammary gland, miR-205 expression was not detectable until the third day of involution with the return of the increased progenitor population. Transplantation of miR-205-deficient mammary epithelial cells revealed that miR-205 is not necessary for mammary gland development but supports stem cell regenerative potential [47]. In a review of miR-205 in breast cancer, its expression has been shown to decrease as breast cancer aggressiveness increases [48]. Indeed, in xenograft mouse models, miR-205 has been shown to reduce tumour growth and vasculogenic recruitment, characteristics of aggressive cancers closer to a stem-like phenotype. Thus, miRNAs enriched in the healthy progenitor cell population may become dysregulated in aggressive breast cancers, imparting a stemlike phenotype.

Epigenetic Regulation
Another mechanism by which cell lineages become restricted, such as from embryonic stem cells to a luminal cell in the developed mammary gland, is through epigenetic regulation. There are two types of epigenetic regulation commonly seen in the mammary gland: DNA methylation and histone modification. Hypomethylation is more commonly found in stem and progenitor cells, and methylation can direct cells into specialized identities. Most studies regarding epigenetic regulation in mammary gland development relate to silencing or activation of genes for lineage-specific differentiation such as luminal-driving GATA3 or stem and basal-driving Angptl2. The role of epigenetic regulation in mammary gland development has been reviewed by Holliday et al. [49]. Although epigenetic regulation in mammary gland development is mainly studied on the coding gene level, miRNAs can promote or be regulated by regulators of methylation or histone modification. For example, the oncogenic miR-150, which is more highly expressed in pregnancy compared to lactation, has been found to repress members of the DNA methyltransferase family DNMT3A and DNMT3B, leading to an increase in the stem-cell-like population, likely due to hypomethylation [50]. Conversely, the puberty-related tumour suppressor miR-184 has been found to be methylated in lymph node metastases samples compared to normal tissue [51]. In puberty, miR-184 is found to be more highly expressed in ductal cells compared to the highly proliferative TEBs and may be identified as an anti-proliferative miRNA. Thus, the function of miRNAs found to be methylated in breast cancer can be examined through the lens of mammary gland development.

Invasion, Migration, and Proliferation
Invasion, migration, and proliferation are regulated through signaling pathways, including the PI3K/Akt/mTOR and Wnt/β-catenin pathways [52,53]. In puberty, the PI3K/Akt pathway can be activated by ligand-induced phosphorylation of fibroblast growth factor receptors or epidermal growth factor receptors, leading to proliferation and cell survival [13]. This pathway is dysregulated in breast cancer, leading to changes in cellular phenotype, metastasis, and drug resistance [52,53]. Dysregulated signaling pathways in puberty and cancer are regulated via miRNA. For example, miR-184 and miR-34a regulate the expression of genes and proteins of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and Wnt pathways, respectively. MiR-184 expression is increased in mature ducts compared to TEBs and reduces the activation of the PI3K/Akt by decreasing phosphorylation of Akt and related genes such as AKT2, PRAS40, and GSK3A. Through deregulation of these genes, tumour proliferation, invasion, and metastatic burden were reduced [51,54]. Similarly, miR-34a was found to reduce Wnt/β-catenin signaling in both puberty and breast cancer, regulating differentiation and suppressing stem-cell like characteristics [45].
During pregnancy, the mammary gland undergoes its final stage of development, once again characterized by significant proliferation and differentiation, leading to the expanded ductal structure that is capable of milk production. The highly proliferative cap cells of the TEB differentiate into myoepithelial cells. Basal and luminal progenitors differentiate into alveolar and ductal cells in pregnancy, which expand and invade the mammary fat pad [13]. The miR-17/92 cluster and miR-21 are both increased during pregnancy relative to early adulthood, indicating it may be involved in the cellular invasion or proliferation requisite for ductal structure growth [55,56]. In breast cancer, the miR-17/92 cluster promotes invasion and metastasis by targeting HBP1, the deactivation of which activates the Wnt/β-catenin pathway. By inhibiting miR-17 in vivo, metastasis of MDA-MB-231 cells was reduced by 50% [57]. In breast cancer, miR-21 is a key miRNA in the promotion of proliferation and dysregulation of apoptosis by inhibiting genes such as PTEN, SMAD7, and PDCD4, ultimately leading to deregulation of the PI3K/Akt/mTOR pathway [58,59]. MiR-21 in MCF-7 cells has also been shown to be modulated by exposure to alpha-linoleic acid in a time-dependent manner, reducing cell viability after 48 h [60]. It would be important to understand if alteration of miRNA regulating invasion, migration, or proliferation pathways during development may be a breast cancer preventative strategy. As well, it would be important to understand if shared miRNAs are altered similarly by dietary or drug interventions during puberty and during breast cancer.

Angiogenesis
During development, angiogenesis is most prominent during pregnancy and lactation during the rapid expansion of the ductal tree. Angiogenesis is necessary for alveolar development and facilitates optimal milk development. In human breast milk, there are high concentrations of vascular endothelial growth factor (VEGF). VEGF is secreted by mammary epithelial cells and mediates vascular growth and permeability during pregnancy and lactation [61]. VEGF has also been implicated in increased angiogenesis in breast cancer and is expressed by tumour endothelial cells. In breast cancer, increased angiogenesis provides nutrients and a platform for migration, leading to tumour growth and metastasis [62]. We found two miRNAs, miR-34 and miR-193b, to be involved in the regulation of angiogenesis in both pubertal and cancer processes. MiR-34 is a tumour suppressive miRNA which has been found to reduce vasculogenic mimicry in breast cancer by targeting AXL tyrosine kinase [63]. Overexpression of miR-34a was also found to reduce invasion and migration. As mentioned previously, miR-34a inhibition led to increased TEB size, an increased progenitor pool, and larger mammary gland in puberty. In puberty, recruitment of vasculature accompanies the rapid proliferation of the TEB. The anti-proliferative effect of miR-34a in puberty corresponds to its anti-angiogenic effect in breast cancer. However, we found no studies confirming that increased angiogenesis contributes to miR-34a-mediated proliferation in puberty.
In pregnancy, miR-193b deletion increased luminal differentiation and proliferation in non-parous and pregnant mice. Yoo et al. postulate that miR-193 mediates proliferation during puberty and pregnancy under the cytokine induced transcription factor STAT5 [64]. STAT5 and prolactin, a key hormone during pregnancy and lactation, have been implicated in a positive autocrine feedback loop which promotes angiogenesis [65]. Thus, the anti-proliferative role of miR-193b is in line with its characterization as a tumour suppressor in breast cancer (Table 1). Mir-193b has been found to reduce vasculogenic mimicry in MDA-MB-231 cells, a triple-negative breast cancer cell line [66]. Other tumour suppressive roles of miR-193 are a reduction in metastasis and drug resistance, which may be mediated by its anti-angiogenic role. Angiogenesis in puberty and pregnancy is partially regulated through miRNA expression, and these miRNAs can allow for identification and further understanding of angiogenic mechanisms which promote metastasis and invasion in breast cancer.

Apoptosis
Apoptosis is an important process for normal breast development. When apoptotic processes are disrupted and reduced, breast cancer arises [67]. Involution is triggered by sustained weaning and returns the mammary gland from a lactating state to its prepregnancy state. It is characterized by a significant increase in apoptosis, breaking down of the basement membrane, expression of metalloproteinases, and recruitment of phagocytes [68]. During involution, the mammary gland regains its pre-pregnancy potential for lactation, including having a carried over and likely newly generated alveolar progenitor population [40]. One miRNA more highly expressed in involution compared to lactation and pregnancy is miR-142-3p. MiR-142-3p targets the 3 UTR of the prolactin receptor (PRLR) mRNA transcript. PRLR is required for the function of prolactin in lobuloalveolar maturation and milk synthesis. MiR-142-3p overexpression increased apoptosis and decreased proliferation in primary murine mammary gland epithelial cells. Downstream, signaling pathways downregulated by miR-142-3p included the apoptosis/protein synthesis-regulating Janus kinase/signal transducer and activator of transcription protein (JAK/STAT) and proliferation-regulating MAPK pathways [69]. In breast cancer, miR-142-3p has a largely tumour-suppressive, anti-proliferative effect (Table 1), increasing apoptotic markers such as the caspase family of cysteine proteases [70][71][72]. Similarly, the miR-424(322)/503 family increases in involution, and knockout of this miRNA reduces acini destruction and apoptosis [73]. Thus, the miR-424(322)/503 family plays an important role in apoptosis and has a tumour-suppressive effect in breast cancer (Table 1). MiR-424-5p has been shown to reduce chemoresistance and decrease breast cancer proliferation by inducing apoptosis and targeting the PI3K/Akt/mTOR pathway [74,75]. By investigating the role of miRNAs in apoptosis, particularly in involution, future studies may identify dysregulated apoptotic miRNAs as potential therapeutic targets.

Conclusions and Future Directions
Since 2006, there have been major strides in understanding the diverse and necessary role of miRNAs as regulators in all biological processes, including mammary gland development. There are clear parallels between processes regulated by miRNAs involved in mammary gland development and breast cancer, with 32 miRNAs so far identified to contribute to both. MiRNAs that stray from their role in development and homeostasis contribute to the development, severity, and prognosis in breast cancer. Processes found to be present in development and aberrant in cancer include EMT, invasion, migration, proliferation, epigenetic regulation, apoptosis, and characteristics such as stemness. By examining miRNAs in development, the mechanisms behind breast cancer incidence, severity, and metastasis can be explained or predicted. From the embryo, miRNAs can be examined for their role in EMT and stemness. During puberty and pregnancy, miRNAs involved in invasion, proliferation, and migration can be investigated. Similarly, the processes of invasion, proliferation, and differentiation can be examined in pregnancy. After lactation, miRNAs involved in the massive apoptosis of the lactation structures can give clues to regulators of apoptosis in breast cancer. Dysregulation of miRNAs in any of these processes can lead to incidence or increased risk of breast cancer. Furthermore, examining miRNAs modified during developmental stages may help to identify and improve the understanding of the function of miRNAs dysregulated in breast cancer. In fact, miRNAs found in development contributing to processes known to be dysregulated in breast cancer can be further investigated.
While studying effect of individual or paired miRNAs in vitro or in vivo is most common, the synergistic effects of miRNA are under explored in breast cancer. During development or homeostasis, miRNAs work in concert to coordinate cellular activity. During mammary gland development, distinct temporal miRNA expression patterns are found during each stage. Further study is required to determine the importance of miRNA synergy during development and their role in breast cancer.
Over a lifetime, miRNAs, genes, and proteins act in concert to regulate development and homeostasis. Understanding these functions are important to better understand how malignancies can arise from their dysregulation. This is especially true for breast cancer, a heterogenous disease which requires consideration of its diverse phenotypes in developing treatments. Since miRNAs can alter mammary gland morphology and breast cancer characteristics, they have immense potential to be nutritional or drug targets for the prevention or treatment of breast cancer.

Mammary Gland Development miRNA Search Strategy
To find miRNAs involved in mammary gland development, PubMed was searched for all original research articles for mammary gland development including animal and human studies. The following search terms were used: "((mammary development) OR (mammary gland development) OR (breast development)) AND (miRNA OR microRNA) NOT (breast cancer)[Title] NOT (tumor[Title]) NOT (carcinoma[Title])". Articles were screened using the Population, Interventions, Comparisons, Outcomes, and Study Designs (PICOS) [245] elements, as outlined in Table 2. Eligibility criteria included primary animal and human studies comparing miRNA expression in 2 or more developmental stages or elucidation of miRNA function in ≥1 developmental stage. Studies examining cell lines, serum, blood, or milk miRNAs were excluded. Data extracted included strain/type/age of animal or human, miRNAs investigated, and the effect of the miRNA on developmental structures or stages.

Breast Cancer miRNA Search Strategy
MiRNAs identified in Section 7.1. (identified as x) were searched in PubMed using the search query "miR-x" AND "breast cancer". The PICOS search strategy is outlined in Table 3. Articles were excluded if only using computational inference.